CHARACTERIZATION OF A 45-KDA FLAVOPROTEIN AND EVIDENCE FOR A RUBREDOXIN, 2 PROTEINS THAT COULD PARTICIPATE IN ELECTRON-TRANSPORT FROM H(2) TO CO2 IN METHANOGENESIS IN METHANOBACTERIUM-THERMOAUTOTROPHICUM

Methanobacterium thermoautotrophicum strains contain a flavoprotein (f lavoprotein A) that copurifies with the H-2:heterodisulfide oxidoreduc tase complex. In this study, we report the iron-dependent synthesis an d biochemical properties of flavoprotein A, cloning and sequencing of the flavoprotein-A-encoding gene (fpaA) and the co-transcription fpaA with two downstream open reading frames, one of which (rdxA) appears t o encode a rubredoxin. Native flavoprotein A has been shown to be a ho modimer of a 45-kDa polypeptide that contains 1.3 mol FMN/45-kDa subun it but no iron or acid-labile sulfur. Catalytic amounts of the H-2:het erodisulfide oxidoreductase complex or of the F-420-reducing hydrogena se reduced flavoprotein A with H-2, at specific rates of 0.3-0.4 U/mg enzyme, generating up to 70% flavin semiquinone before reduction to th e flavin hydroquinone was observed. This intermediate accumulation of the semiquinone species had a kinetic rather than a thermodynamic basi s, because the semiquinone form of flavoprotein A, generated by photor eduction, disproportionated quantitatively to the quinone and hydroqui none species, The midpoint potential of the quinone/hydroquinone coupl e was estimated to be 230 +/- 15 mV, at pH 7.6, versus the normal hydr ogen electrode. Quantitation of Western blots demonstrated that flavop rotein A constituted approximately 1.5% of the soluble protein in cell s grown in an iron-sufficient medium but that this increased to about 6% of the cellular protein when the iron the medium was depleted. The increase in the flavoprotein A content of cells grown under iron-limit ing conditions was mirrored by a decrease in the content of the iron-r ich polyferredoxin that also copurified with the H-2:heterodisulfide o xidoreductase complex. The fpaA gene, cloned and sequenced from M. the moautotrophicum strain Delta H, encodes 404 amino acids in a sequence that has a C-terminal domain (approximately 130 amino acid residues) w ith features consistent with a flavodoxin structure. The remainder of flavoprotein A has sequences that are also predicted to be present in the N-terminal region of the orf14 gene product, which also appears to be an enlarged flavodoxin, encoded in the nif region of Rhodobacter c apsulatus. Immediately downstream from fpaA, two open reading frames d esignated olfX and rdxA, have been located and shown by Northern-blot analyses to be co-transcribed with fpaA, although approximately 50% of fpaA-orfX-rdxA transcripts terminated or were cleaved within rdxA. Pr imer extension studies revealed that transcription of this transcripti onal unit (the fpa operon) was initiated 32 nucleotides upstream of fp a4, at a site 25 nucleotides downstream from a sequence consistent wit h an archaeal TATA-box promoter element. rdxA is predicted to encode a 19.6-kDa polypeptide that is most closely related to rubredoxin I of Pseudomonas oleovorans, and that contains an N-terminal motif with fea tures found in all rubredoxins, including the four cysteine residues t hat coordinate the iron atom that provides the redox center of rubredo xins.